Miniminally invasive dermal electroporation device

ABSTRACT

The disclosure is directed to a device for electroporating and delivering one or more antigens and a method of electroporating and delivering one or more antigens to cells of epidermal tissues using the device. The device comprises a housing, a plurality of electrode arrays projecting from the housing, each electrode array including at least one electrode, a pulse generator electrically coupled to the electrodes, a programmable microcontroller electrically coupled to the pulse generator, and an electrical power source coupled to the pulse generator and the microcontroller. The electrode arrays define spatially separate sites.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/502,198, filed Jun. 28, 2011, the content of which is incorporatedherein by reference in its entirety.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

Activities relating to the development of the subject matter of thisinvention were funded at least in part by U.S. Government, Army ContractNo. W81XWH-11-C-0051, and thus the U.S. may have certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to an electroporation device that iscapable of delivering one or more plasmid vaccines simultaneously atspatially separate sites in a tolerable manner.

BACKGROUND

A major obstacle to effective vaccination via antigenic plasmids is theneed of the DNA vaccine to be delivered intracellularly. The delivery ofnaked DNA through a standard intramuscular injection is notoriouslyinefficient outside of rodent models. Historically, this has led to aninability to achieve robust immune responses in large mammals andhumans. Several strategies have been developed to enhance the expressionof DNA-based vaccines, such as codon-optimization, RNA optimization,leader sequence addition and the development of optimized consensussequences. These optimization strategies can lead to improved,cross-reactive immune responses. The addition of co-delivered gene-basedmolecular adjuvants is another area where an augmentation of resultingimmune responses frequently occurs. Despite the improvements in vectordesign and use of molecular adjuvants, there is still a clear need foran efficient method of administration of DNA vaccines that results inhigh level expression of the plasmid in the desired cell type of thedesired tissue, most commonly, muscle, tumor or skin.

Drug delivery to dermal tissue (intradermal) is an attractive method ina clinical setting for a number of reasons. The skin is the largestorgan of the human body, the most accessible, and easily monitored, aswell as being highly immuno-competent. However, the impervious, barrierfunction of the skin has been a major obstacle to efficient trans-dermaldrug delivery.

Human skin comprises approximately 2 m² in area and is around 2.5 mmthick on average, making it the largest organ of the human body.Conventionally, the skin has two broad tissue types, the epidermis andthe dermis. The epidermis is a continually keratinizing stratifiedepithelium. The outermost layer of skin is the stratum corneum (SC) andfunctions as the primary barrier. The SC is a 15-30 cell thick layer ofnon-viable but biochemically active corneocytes. The other three strataof the epidermis (S. granulosum, S. spinosum, S. basale) all containketatinocytes at different stages of differentiation as well as theimmune Langerhans cells and dermal dendritic cells.

Both physical and chemical methods for trans-dermal drug delivery andgene delivery have been detailed by groups worldwide. Iontophoresis,lipid delivery and gene gun are such examples. A physical method totemporarily increase skin permeability is electroporation (“EP”).Electroporation involves the application of brief electrical pulses thatresult in the creation of aqueous pathways within the lipid bi-layermembranes of mammalian cells. This allows the passage of largemolecules, including DNA, through the cell membrane which wouldotherwise be less permeable. As such, electroporation increases theuptake or the extent to which drugs and DNA are delivered to theirtarget tissue.

Although the precise mechanism by which electroporation enables celltransformation has not been elucidated, a proposed theoretical modelinvolves a poration event due to the destabilization of the membrane,followed by the electrophoretic movement of charged molecules into thecell. For electroporation to occur, the formation of pores requires thata threshold energy be achieved and the movement produced by theelectrophoretic effect depends upon both the electric field and thepulse length.

In the case of DNA vaccines, electroporation has been shown toquantitatively enhance immune responses, increase the breadth of thoseimmune responses as well as improve the efficiency of dose. Morerecently, the DNA-EP platform has been successfully translated into thehuman clinical setting and has demonstrated significantly improvedimmune responses in several vaccine studies. Therefore, there hasdeveloped a need for a dermal electroporation device that would beconsidered tolerable, user-friendly and easily amenable to massproduction, while continuing to achieve high transfection ratesresulting in robust immune responses.

Although a number of intramuscular devices have now successfully enteredclinical trials, the procedure is generally considered invasive andpainful. To be considered amenable to mass vaccination, especially in apediatric setting, a solution for a more tolerable electroporationmethod is needed. Accordingly, an effective dermal electroporationdevice that is capable of delivering a multi-agent DNA vaccine in atolerable manner is desirable.

SUMMARY OF THE INVENTION

The present disclosure is directed to a device for electroporating anddelivering one or more antigens. The device comprises a housing, aplurality of electrode arrays projecting from the housing, eachelectrode array including at least one electrode, a pulse generatorelectrically coupled to the electrodes, a programmable microcontrollerelectrically coupled to the pulse generator, and an electrical powersource coupled to the pulse generator and the microcontroller. Theelectrode arrays define spatially separate sites. The electrodes areconfigured to deliver an electroporating pulse to cells of epidermaltissues. The microcontroller is configured to adjust parameters of theelectroporating pulse of each electrode array independently.

The present disclosure is also directed to a method of electroporatingand delivering one or more antigens to cells of epidermal tissues usingthe device described herein. In embodiments, the antigens generallyinclude DNA vaccine plasmids, peptides, small molecules, andcombinations thereof. The method comprises administering the one or moreantigens to the cells of the epidermal tissues, contacting the epidermaltissues with the electrodes, and delivering the electroporating pulses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A), 1(B), and 1(C) are perspective views of a minimally invasivedevice (MID) for EP according to an embodiment.

FIG. 2 is a schematic illustration of an electrical system of theembodiment of FIGS. 1(A), 1(B), and 1(C).

FIGS. 3(A), 3(B), and 3(C) are perspective views of a MID for EPaccording to another embodiment.

FIG. 4 is a schematic illustration of an electrical system of theembodiment of FIGS. 3(A), 3(B), and 3(C).

FIGS. 5(A), 5(B), and 5(C) are fluorescent micrographs of greenfluorescent protein (GFP) expression following injection andelectroporation of plasmids using an MID with (A) stainless steelelectrodes or (B) gold electrodes or with (C) injection only and noelectroporation as a control. GFP pixel intensity was calculated (D).

FIGS. 6(A) and 6(B) are views of a MID for EP as (A) a side view of a 1mm-spaced array (top) and 1.5 mm-spaced array (bottom) electrode handpiece, and as (B) a close up of the face of a 1 mm-spaced array showingall 25 needle electrodes.

FIGS. 7(A), 7(B), and 7(C) are fluorescent micrographs of GFP expressionfollowing injection and electroporation of plasmids using an MID with1.5 mm or 1 mm electrode spacing and (A) stainless steel electrodes or(B) gold electrodes. A set with injection only and no electroporationwas used as a control. GFP pixel intensity was calculated (C).

FIG. 8 shows graphs of current and resistance for MIDs with 1.5 mm or 1mm electrode spacing. Electrodes were either gold or stainless steel(SS) in composition.

FIG. 9 shows graphs of current and impedence for MIDs with 1.5 mm or 1mm electrode spacing at 5 or 15 volts.

FIG. 10(A) shows fluorescent micrographs of GFP expression followinginjection and electroporation of plasmids using an MID with 1.5 mm or 1mm electrode spacing at 5 or 15 volts. GFP pixel intensity wascalculated (B).

FIG. 11(A) shows fluorescent micrographs of GFP expression followinginjection and electroporation of different concentrations (0.5, 0.25,and 0.1 mg/mL) of plasmid using an MID with 1.5 mm or 1 mm electrodespacing at 15 volts. GFP pixel intensity was calculated (B).

FIGS. 12(A), 12(B), and 12(C) are fluorescent micrographs of lymphocytestaining following injection and electroporation of plasmids using anMID. FIG. 12(A) is a skin biopsy in an untreated animal at 20×magnification. FIG. 12(B) is a skin biopsy from an animal treated withplasmid expression GFP at 20× magnification. FIG. 12(C) is the sample inFIG. 12(B) but at 40× magnification.

FIG. 13(A) is a perspective view of a MID for EP according to anembodiment, FIG. 13(B) is a photograph of an MID with a 4×4 array and1.5 mm spacing, FIG. 13(C) is a photograph of an MID with a 5×5 arrayand 1.0 mm spacing, and FIG. 13(D) is a photograph showing a side byside comparison of the MIDs in FIGS. 13(B) and 13(C).

FIG. 14 is a fluorescent micrograph showing the GFP expression followingan intradermal administration of a reporter gene plasmid and EP with thedual-head MIDs of FIG. 13(B), compared to the GFP expression followingthe intradermal injection alone (no EP).

DETAILED DESCRIPTION

The present invention is directed to an electroporation device that canprovide heterogeneous intradermal delivery of antigens to a mammal. Oneor more antigens can be delivered simultaneously at spatially separatedsites in a tolerable manner via a minimally invasive device (MID) havinga plurality of electrode arrays. The electrode arrays are configured tovary the electroporating pulse from array to array. For example, eachelectrode array can be independently and selectively activated orcontrolled. Thus, the MID enables a heterogeneous delivery of antigens.Dermal electroporation via this MID reflects a clinically acceptablemethod to effectively deliver vaccines to the skin of a subject. Thisdevice is amenable to delivering multiple vaccines in multiple forms(nucleic acid, protein, small molecules, or a combination thereof)simultaneously while removing potential concerns withimmune-interference resulting from the co-delivery of multiple antigens.It also allows the ability to deliver higher doses of a single antigenduring a single treatment.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “and” and“the” include plural references unless the context clearly dictatesotherwise.

For the recitation of numeric ranges herein, each intervening numbertherebetween with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

The term “electroporation” as used herein refers to the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; the pores'presence allows biomolecules suchas plasmids, oligonucleotides, siRNA, drugs, ions, and water totemporarily pass from one side of the cellular membrane to the other.

The term “minimally invasive” as used herein refers to a limitedpenetration by the needle electrodes of the provided EP device, and caninclude noninvasive electrodes (or nonpenetrating needles). Preferably,the penetration is to a degree that penetrates through stratum corneum,and preferably enters into the outermost living tissue layer, thestratum granulosum, but does not penetrate the basal layer. Thepenetration depth preferably does not exceed 0.1 mm, and in someembodiments the penetration depth ranges from about 0.01 mm to about0.04 mm to break through stratum corneum. This can be accomplished usingan electrode that has a trocar end ground to provide a sharp point thatallows penetration through the stratum corneum but avoids a deeperpenetration.

The terms “tolerable” and “nearly painless” are used interchangeablyherein, and when referring to EP, mean a substantially lower level ofpain associated with EP than with typically available EP devices. Morespecifically, a tolerable or near painless EP is the result ofcombination of using the device described herein, avoiding EP of muscle,along with delivering low electrical fields to the epidermal layersbetween the stratum corneum and the basal layers. Preferably theelectrical fields will comprise low voltage levels, for example from0.01 V to 70 V, or from 1 V to 15 V. When measured using a visual analogscale, subjects experiencing EP with the device described hereinaccording to the methods provided herein experience pain levels that arewithin 20% (of the full scale) from their painless or pain free score,or for example, within 2 points, with 0-10 full scale, and preferablywithin 10% from their painless score.

The term “substantially prevents damage” is used herein to refer to anamount of energy that is delivered by the described devices to thetarget cells to electroporate said cells and cause minimal discernibledamage to same cells. Preferably, there is no discernable macroscopichistological damage or alteration to such cells.

2. MINIMALLY INVASIVE DEVICE

The present invention is directed to a minimally invasive device (MID)having a plurality of electrode arrays that are configured to vary theelectroporating pulse from array to array. FIGS. 1(A), 1(B), and 1(C)disclose an MID 100 having a plurality of electrode arrays forelectroporating and delivering one or more antigens. The MID 100comprises a housing 102 having a tip portion 104, a plurality ofelectrode arrays 106, 108, coupled to the tip portion 104, eachelectrode array 106, 108 including electrodes 110 arranged in a square4×4 pattern. In some embodiments, one or both of the tip portion 104 andthe electrodes 110 can be detached from the rest of the MID 100, e.g.,for sterilizing after use so that the detached parts can be used again.Alternatively, one or both of the tip portion 104 and the electrodes 110can be for a single use.

The electrode arrays 106, 108 define spatially separate sites. AlthoughFIGS. 1(A), 1(B), and 1(C) illustrate the MID 100 as including twoelectrode arrays 106, 108, in other embodiments the MID 100 can includemore than two electrode arrays, e.g., three or more, four or more, fiveor more, six or more, seven or more, eight or more, nine or more, or tenor more electrode arrays.

In some embodiments, each array 106, 108 can include a 4×4 array ofelectrodes 110 having a respective length of 0.1 mm, 0.2 mm, 0.3 mm, 0.4mm, 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5mm, 5 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, or 10.0 mm. Although in theillustrated embodiment each array 106, 108 includes a 4×4 array ofelectrodes 110, i.e., 16 electrodes 110, in other embodiments, each ofthe arrays 106, 108 can respectively include other numbers and/orpatterns of electrodes 110. For example, each of the arrays 106, 108 canrespectively include electrodes 110 arranged in a pattern of 1×1, 1×2,1×3, 1×4, 1×5, 1×6, 1×7, 1×8, 1×9, 1×10, 2×1, 2×2, 2×3, 2×4, 2×5, 2×6,2×7, 2×8, 2×9, 2×10, 3×1, 3×2, 3×3, 3×4, 3×5, 3×6, 3×7, 3×8, 3×9, 3×10,4×1, 4×2, 4×3, 4×4, 4×5, 4×6, 4×7, 4×8, 4×9, 4×10, 5×1, 5×2, 5×3, 5×4,5×5, 5×6, 5×7, 5×8, 5×9, 5×10, 6×1, 6×2, 6×3, 6×4, 6×5, 6×6, 6×7, 6×8,6×9, 6×10, 7×1, 7×2, 7×3, 7×4, 7×5, 7×6, 7×7, 7×8, 7×9, 7×10, 8×1, 8×2,8×3, 8×4, 8×5, 8×6, 8×7, 8×8, 8×9, 8×10, 9×1, 9×2, 9×3, 9×4, 9×5, 9×6,9×7, 9×8, 9×9, 9×10, 10×1, 10×2, 10×3, 10×4, 10×5, 10×6, 10×7, 10×8,10×9, 10×10, or multiples of 11-100 and any combination thereof. Thepatterns can be arranged in various shapes such as squares, triangles,rectangles, parallelograms, circles or any other geometric shape. Theneedle-shaped electrodes 110 can comprise gold, platinum, titanium,stainless steel, aluminum, or any other conductive metal. The electrodescan be coated or plated with a metal such as gold, copper, platinum,silver, or any other conductive metal.

In some embodiments, each electrode 110 is needle-shaped. That is, theelectrodes 110 each include a shaft 112 and a tapered tissue-penetratingor trocar end 114. Although FIG. 2(B) illustrates the electrodes 110 asbeing generally cylindrical, in other embodiments, at least one of theelectrodes 110 can assume any geometric form, including, but not limitedto, a semi-cylindrical, a regular polyhedral, and an irregularpolyhedral shape, derivatives thereof, and combinations thereof. Thetissue-penetrating end 114 can facilitate the electrodes 110 penetratingthrough the stratum corneum and reaching the stratum granulosum. In someembodiments, the tissue-penetrating end 114 allows the electrode 110 topenetrate through the stratum corneum but avoids deep penetration. Tothis end, the tissue-penetrating end 114 can have a length of about 0.1mm or less, or about 0.01 mm to about 0.04 mm.

The illustrated electrodes 110 are configured to deliver anelectroporating pulse to cells of epidermal tissues. In someembodiments, the electroporating pulses are associated with anelectrical field that substantially prevents damage in the cells of theepidermal tissues. In further embodiments, the electroporating pulsesare associated with an electrical potential that is nearly painless asmeasured by a visual analog scale. The visual analog scale isessentially a 100-mm-long horizontal line on which 0 mm indicates nopain and 100 mm indicates the worst pain. Near painless is a score usingthe visual analog scale that produces a mean score of about 20 mm orless (within a 95% confidence interval), and preferably 10 mm or less(within a 95% confidence interval).

In some embodiments, adjoining electrodes 110 are spaced apart from oneanother at a distance of no more than about 1.5 mm. In furtherembodiments, adjoining electrodes 110 are spaced apart from one anotherat a distance of no more than about 1.0 mm. A shorter distance betweenthe electrodes 110 means that the electrodes 110 are packed in a morecompact manner, which can increase the efficacy of the MID 100 andtherefore can be desirable. In some embodiments, each electrode 110 canbe spaced apart from each adjacent electrode 110 at a distance of 150 mmor less, from 100 mm to 1.0 mm, from 50 mm to 1.0 mm, from 40 mm to 1.0mm, from 30 mm to 1.0 mm, from 20 mm to 1.0 mm, from 10 mm to 1.0 mm,from 5.0 mm to 2.0 mm, from 5.0 mm to 1.0 mm, approximately 2.0 mm,approximately 1.5 mm, or approximately 1.0 mm.

Referring also to FIG. 2, pulse generators 116, 118 are electricallycoupled to respective electrode arrays 106, 108. In some embodiments, atleast one of the pulse generators 116, 118 can be the Elgen 1000 (InovioPharmaceuticals, Inc., Blue Bell, Pa.) pulse generator (not shown). Inother embodiments, however, the electroporating pulse can be generatedusing other suitable mechanisms. In the illustrated embodiment, aprogrammable microcontroller 120 is electrically coupled to the pulsegenerators 116, 118. In response to an input condition/signal, themicrocontroller 120 is capable of adjusting EP parameters of eachelectrode array 106, 108 independently, depending on the usagerequirements or preferences for each electrode array 106, 108. Thus, themicrocontroller 120 is configured to vary the electroporating pulse fromarray to array. For example, the pulse voltage, current, duration, andquantity of the applied electrical pulses can be varied from array toarray so as to vary the Joules per cm³ applied at each injection site.In some embodiments, the microcontroller 120 is configured to deliverthe electroporating pulses substantially simultaneously. In theillustrated embodiment, each electrode array 106, 108 is driven with arespective pulse generator 116, 118. The MID 100 also includes anelectrical power source 122 coupled to the pulse generators 116, 118 andthe microcontroller 120 for providing electrical power. In theillustrated embodiment, the electrical power source 122 is a high andlow voltage supply, although other power sources performing the samefunction as the electrical power source 122 disclosed herein can be usedinstead.

In some embodiments, the electroporating pulse of each electrode array106, 108 is associated with an electrical potential of 0.01 V to 70 V,0.01 V to 50 V, 0.01 V to 40 V, 0.01 V to 30 V, 0.01 V to 20 V, 0.01 Vto 15 V, 0.1 V to 70 V, 0.1 V to 50 V, 0.1 V to 40 V, 0.1 V to 30 V, 0.1V to 20 V, 0.1 V to 15 V, 1V to 30V, 1V to 20V, 1 V to 15 V, 15V to 30V,or 15V to 30V. In further embodiments, the electrical potential ispreferably low so that the EP is tolerable or nearly painless asmeasured by a visual analog scale, yet sufficiently high so as to effecttransfection of the cells in the epidermal tissues. For example, theelectrical potential can be 5V, 10V, 15 V, or in some embodiments 20V,when adjacent electrodes 110 of the MID 100 are spaced apart by 1.0 mmto 2.0 mm.

In some embodiments, each electroporating pulse of each electrode array106, 108 is associated with an electrical current of 0.1 mA to 100 mA,0.2 mA to 100 mA, 0.5 mA to 100 mA, 1 mA to 100 mA, 1 mA to 80 mA, 1 mAto 60 mA, 1 mA to 50 mA, 1 mA to 40 mA, 1 mA to 30 mA, 10 mA to 50 mA,10 mA to 40 mA, 10 mA to 30 mA, 10 mA to 20 mA, or 10 mA to 15 mA, orapproximately 10 mA, or in some embodiments approximately 20 mA. Likethe electrical potential, the electrical current is preferably low sothat the EP is tolerable or nearly painless as measured by a visualanalog scale, yet sufficiently high so as to effect transfection of thecells in the epidermal tissues.

In some embodiments, the electroporating pulse of each electrode array106, 108 is associated with a duration of from 5 ms to 250 ms, 10 ms to250 ms, 20 ms to 250 ms, 40 ms to 250 ms, 60 ms, to 250 ms, 80 ms to 250ms, 100 ms to 250 ms, 20 ms to 150 ms, 40 ms to 150 ms, 60 ms to 150 ms,80 ms to 150 ms, 100 ms to 150 ms, 100 ms to 140 ms, 100 ms to 130 ms,100 ms to 120 ms, 100 ms to 110 ms, or approximately 100 ms. In someembodiments, the duration is preferably short so that the EP istolerably or nearly painless as measured by a visual analog scale, yetsufficiently long so as to effect transfection of the cells in theepidermal tissues.

In some embodiments, the microcontroller 120 is configured to adjust arespective quantity of electroporating pulses for each electrode array106, 108 independently, and the number of electrical pulses can be 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more.By increasing the quantity of electroporating pulses and reducing theenergy per pulse, the amount of pain perceived or experienced by asubject can also be reduced as compared with fewer pulses at higherenergy. Preferably, a lower number of pulses, which does not reduceimmune response that is generate, is used as it results in less painexperienced by the subject. Furthermore, less pain and bettertolerability results by using lower energy per pulse. In someembodiments, the quantity of electroporating pulses is about 1 to about10, preferably about 1 to about 10, and more preferably about 1 to about3. In some embodiments 3 pulses are used.

In some embodiments, the electrode arrays 106, 108 are spaced apart fromone another at least by a distance so as to substantially preventinterference of the two antigens delivered by the two arrays 106, 108when the electroporating pulses are delivered. Plasmid interference hasbeen observed for a number of antigens when they are deliveredsequentially at the same site in the skin. Though not wishing to bebound by a particular theory, this could be due to interference ateither the transcriptional level (possible competition at the promoter,etc.) or the translational level (mis-folding or dimerization at theprotein level). The MID 100 having a plurality of spaced electrodearrays 106, 108 could eliminate this interference effect, which cannegatively affect the resulting immune response. Moreover, the MID 100having a plurality of electrode arrays 106, 108 could negate the needfor two separate treatments, allowing a treated subject to experienceone incident of treatment, thus reducing the pain that is experienced.

In some embodiments, the MID 100 includes switches (not shown)electrically coupled to each electrode array 106, 108 for selectivelyactivating each electrode array 106, 108. For example, the MID 100 canbe triggered by a foot pedal or a trigger button, or any other triggerconnected to an electrical circuit.

FIGS. 3 and 4 illustrate an MID 200 including a pulse generator 202according to another embodiment. This embodiment employs much of thesame structure and has many of the same properties as the embodiment ofthe MID 100 described above in connection with FIGS. 1(A)-1(C).Accordingly, the following description focuses primarily upon thestructure and features that are different than the embodiment describedabove in connection with FIGS. 1(A)-1(C). Structure and features of theembodiment shown in FIGS. 3 and 4 that correspond to structure andfeatures of the embodiment of FIGS. 1(A)-1(C) are designated hereinafterwith like reference numbers.

In this embodiment, the pulse generator 202 is powered by a battery 204.In the illustrated embodiment, the battery 204 is within the housing102. As such, the MID 200 can be portable. The battery 204 can be alithium ion, nickel metal hydride, lead acid, or nickel cadmium battery.

Referring to FIG. 4, the pulse generator 202 is a high and low voltagedriver. The pulse generator 202 in this embodiment drives both electrodearrays 106, 108. A microcontroller 206 is electrically coupled to thepulse generator 202. The microcontroller 206 is capable of adjusting theelectroporation parameters of each electrode array 106, 108independently for example in response to an input condition/signal. Thepulse generator 202 can generate pulses with EP parameters as adjustedby the microcontroller 206, and, in cooperation with the battery 204,amplify the generated pulses as needed.

3. METHOD OF ELECTROPORATING AND DELIVERING ONE OR MORE ANTIGENS

In an aspect, the MID 100, 200 having a plurality of electrode arrays106, 108 described herein can be used in a method of electroporating anddelivering one or more antigens, as discussed below, through the skin,organs, or other body parts of a subject. That is, the MID 100, 200 canbe used to apply a transmembrane electric field pulse that inducesmicroscopic pathways (pores) in a bio-membrane, allowing the delivery ofone or more antigens from one side of the cellular membrane to theother. The method can comprise the steps of administering the antigen tothe cells of the epidermal tissues, contacting the epidermal tissueswith the electrodes, and delivering an electroporating pulse to generatean immune response. The method can further comprise simultaneouslydelivering antigen to the cells and delivering an electroporating pulseto generate an immune response.

Administering the Antigen to the Cells of the Epidermal Tissues

A plurality of antigens is first injected intradermally at spatiallyseparated sites. In some embodiments, the antigen is intradermallydelivered to the target tissue using the Mantoux technique, e.g., usinga 29 gauge injection needle.

Contacting the Epidermal Tissues with the Electrodes

Next, the epidermal tissues are penetrated with at least one electrode110 at a depth of about 0.1 mm or less, or about 0.01 mm to about 0.04mm. The injection sites and the tissue-penetration sites are preferablyco-localized. in some examples, to facilitate co-localizing or centeringthe injection sites and the tissue-penetration sites, the epidermaltissues cancan be marked or indented before the intradermal injection.

Delivering an Electroporating Pulse

Once the epidermal tissues are penetrated, the epidermal tissues arecontacted with the electrodes 110 and an electroporating pulse isdelivered. In some embodiments, the electroporating pulses areassociated with an electrical field that substantially prevents damageto the cells of the epidermal tissues. In further embodiments, theelectroporating pulses are associated with an electrical potential thatis nearly painless as measured by a visual analog scale. For example,the electroporating pulses are associated with an electrical potentialof about 1 volts to about 30 volts, or preferably about 15 volts toabout 20 volts, an electrical current of about 1 mA to about 50 mA, orpreferably about 10 mA to about 15 mA, and a duration ranging from about80 ms to about 150 ms, or preferably 100 ms, or a combination thereof.These pulses can be delivered in a series, preferably 1-10 pulses, andmore preferably 1-3 pulses.

A drawback to conventional intradermal delivery is a limitation to thevolume that can be delivered to the skin. For a single needle injection,generally volumes no larger than 100-150 μl can be delivered directly tothe skin due to issues with dermal delamination. Because the antigen canonly be produced at concentrations not in excess of 10 mg/mL, the volumelimitation can constrain the resulting dose.

In some embodiments, the MID 100, 200 can preferably deliver higherdoses of a single vaccine. The use of the MID 100, 200 avoids the singleinjection volume limitation of 100-150 μL. In some embodiments,significantly higher doses can be delivered simultaneously with a singletreatment without any added discomfort to the patient. The ability todeliver higher doses could have significant positive effects on theresulting immune responses for specific vaccines. The use of amulti-head device also has the added benefit of directly targeting morecells than a single array device. Increased numbers of cells transfectedwith an antigen could result in improved immune responses throughincreased presentation to antigen presenting cells.

In some embodiments, the disclosed method can be administered to asubject such as a mammal. The mammal can be a human, monkey, dog, cat,livestock, guinea pig, mouse, or a rat. The livestock can be bovine, apig, a sheep, or a cow, for example.

4. ANTIGEN

The present invention is also directed to methods of delivering at leastone antigen using the MID 100, 200 having a plurality of electrodearrays 106, 108, as discussed above. The method can be directed todelivery of two or more antigens or a combination thereof usingheterogeneous delivery by the MID 100, 200. In certain embodiments, theMID 100, 200 described herein can be used to enhance delivery of anantigen. As used herein, “antigen” refers to any substance or organismthat provokes an immune response (produces immunity) when introducedinto the body.

In some embodiments, the antigen can be derived from an infectious agentor a self-antigen, e.g., a prostate cancer antigen such asprostate-specific antigen (PSA) or prostate-specific membrane antigen(PSMA). The particular antigen used is not critical. Antigens are knownin the art and can be incorporated for use in the methods andcompositions provided herein using any common method. Non-limiting listsof suitable antigens for use in the various aspects and embodimentsdescribed herein can be found in the literature, for example, BioCarbChemicals Catalogue; and The Jordan Report: Accelerated Development ofVaccine 1995 NIH, Bethesda, Md., 1995, both of which are incorporatedherein by reference. Antigens can include, but are not limited to,nucleic acids, peptides, small molecules, chemotherapeutics,immunotherapeutics, or combinations thereof. An antigen can include animmunogen.

In some embodiments, the antigen comprises a nucleic acid. Nucleic acidrefers to a polynucleotide compound, which includes oligonucleotides,comprising nucleosides or nucleoside analogs that have nitrogenousheterocyclic bases or base analogs, covalently linked by standardphosphodiester bonds or other linkages. Nucleic acids can include RNA,DNA, chimeric DNA-RNA polymers, or analogs thereof. The DNA can be aplasmid expressing a particular antigen of interest. For example, theplasmid can be a SynCon influenza construct (Inovio Pharmaceuticals,Inc., Blue Bell Pa.).

In some embodiments, the antigen comprises a peptide. Peptides includeany amino acid sequence. Peptides can be synthetic or isolated from anatural source. The peptide can be a protein. The peptide can be anantibody or antibody fragment.

In some embodiments, the antigen comprises a small molecule. Smallmolecules include organic and inorganic compounds.

In some embodiments the antigen comprises a chemotherapeutic.Chemotherapeutics can include cytotoxic or cytostatic drugs such as, forexample, methotrexate (amethopterin), doxorubicin (adrimycin),daunorubicin, cytosinarabinoside, etoposide, 5-fluorouracil, melphalan,chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide),cis-platinum, vindesine (and other vinca alkaloids), mitomycin,bleomycin, purothionin (barley flour oligopeptide), macromomycin.1,4-benzoquinone derivatives, and trenimon.

In some embodiments, the antigen includes a cytokine. Cytokine refers toa substance secreted by cells of the immune system that carry signalslocally between cells. Cytokines include proteins, peptides, andglycoproteins. Cytokines include, but are not limited to, interferons,chemokines, TGF-β, TNF-α, and interleukins. Interleukins include IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,IL-32, IL-33, IL-35, and IL-36. Cytokines can be derived from a humansource or a transgenic non-human source expressing a human gene.

Antigens can include, but are not limited to, microbial antigens such asparasitic antigens, viral antigens, bacterial antigens, fungal antigens,cancer antigens, vaccine antigen additive drugs such as cocaine andnicotine derivatives, attenuated or killed bacteria, attenuated orkilled virus, autoimmune antigens, or nonstructural protein antigens, orany combination thereof. In some embodiments, the antigen comprises atleast one flu, autoimmune, cocaine, or cancer antigen.

In some embodiments an antigen comprises any antigen derived frombacterial surface polysaccharides which can be used incarbohydrate-based vaccines. Bacteria typically express carbohydrates onthe cell surface as part of glycoproteins, glycoplipids, O-specific sidechains of lipopolysaccharides, capsular polysaccharides and the like.Non-limiting examples of suitable bacterial strains includeStreptococcus pneumonia, Neisseria meningitidis, Haemophilus influenza,Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., andGroup B streptococci. In some embodiments any known bacterialcarbohydrate epitope (e.g., those described in Sanders, et al. Pediatr.Res. 1995, 37, 812-819; Bartoloni, et al. Vaccine 1995, 13, 463-470;Pirofski, et al., Infect. Immun. 1995, 63, 2906-2911; U.S. Pat. No.6,413,935; and International Publication No. WO 93/21948) can be used asan antigen in the compositions and methods herein described.

Some embodiments provide for an antigen that comprises a viral antigen.Non-limiting examples of viral antigens include those derived from HIV(e.g., gp120, nef, tat, pol), influenza, and West Nile Virus (WNV). Insome embodiments, the antigen can comprise whole killed virus orattenuated virus.

Some aspects provide for a fungal antigen. Non-limiting examples offungal antigens include those derived from Candida albicans,Cryptococcus neoformans, Coccidoides spp., Histoplasma spp., andAspergillus spp.

Some embodiments provide for an antigen derived from a parasite.Non-limiting examples of parasitic antigens include those derived fromPlasmodium spp., Trypanosoma spp., Schistosoma spp., Leishmania spp. andthe like.

In some embodiments the antigen comprises a carbohydrate epitope.Non-limiting examples of carbohydrate epitopes that can be used in theaspects and embodiments described herein include: Galα1,4Galβ (forbacterial vaccines); GalNAcα (for cancer vaccines);Manβ1,2(Manβ)_(n)Manβ-(for fungal vaccines useful against, for example,C. albicans), wherein n is any integer, including zero;GalNAcβ1,4(NeuAcα2,3)Galβ1,4Glcβ-O-ceramide (for cancer vaccines);Galα1,2(Tyvα1,3)Manα1,4Rhaα1,3Galα1,2-(Tyα1,3)Manα4Rha- andGalα1,2(Abeα1,3)Manα1,4Rhaα1,3Galα1,2(Abeα1,3)Manα1,4Rhaα1,3Galα1,2(Abeα1,3)Manα1,4Rha (both of which are usefulagainst, for example, Salmonella spp.). Description of other exemplarycarbohydrate epitopes as antigens or immunogens and the synthesisthereof are described further in U.S. Pat. No. 6,413,935, incorporatedherein by reference.

Other examples of antigens include, but are not limited to, those thatproduce an immune response or antigenic response to the followingdiseases and disease-causing agents: anthrax; adenoviruses; Bordetellapertussus; Botulism; bovine rhinotracheitis; Branhamella catarrhalis;canine hepatitis; canine distemper; Chlamydiae; Cholera;coccidiomycosis; cowpox; cytomegalovirus; cytomegalovirus; Dengue fever;dengue toxoplasmosis; Diphtheria; encephalitis; EnterotoxigenicEscherichia coli; Epstein Barr virus; equine encephalitis; equineinfectious anemia; equine influenza; equine pneumonia; equinerhinovirus; feline leukemia; flavivirus; Globulin; Haemophilus influenzatype b; Haemophilus influenzae; Haemophilus pertussis; Helicobacterpylori; Hemophilus spp.; hepatitis; hepatitis A; hepatitis B; hepatitisC; herpes viruses; HIV; HIV-1 viruses; HIV-2 viruses; HTLV; Influenza;Japanese encephalitis; Klebsiellae spp. Legionella pneumophila;leishmania; leprosy; lyme disease; malaria immunogen; measles;meningitis; meningococcal; Meningococcal Polysaccharide Group A,Meningococcal Polysaccharide Group C; mumps; Mumps Virus; mycobacteria;Mycobacterium tuberculosis; Neisseria spp; Neisseria gonorrhoeae;Neisseria meningitidis; ovine blue tongue; ovine encephalitis;papilloma; parainfluenza; paramyxovirus; paramyxoviruses; Pertussis;Plague; Pneumococcus spp.; Pneumocystis carinii; Pneumonia; Poliovirus;Proteus species; Pseudomonas aeruginosa; rabies; respiratory syncytialvirus; rotavirus; Rubella; Salmonellae; schistosomiasis; Shigellae;simian immunodeficiency virus; Smallpox; Staphylococcus aureus;Staphylococcus spp.; Streptococcus pneumoniae; Streptococcus pyogenes;Streptococcus spp.; swine influenza; tetanus; Treponema pallidum;Typhoid; Vaccinia; varicella-zoster virus; and Vibrio cholerae. Theantigens or immunogens can include various toxoids, viral antigensand/or bacterial antigens such as antigens commonly employed in thefollowing vaccines: chickenpox vaccine; diphtheria, tetanus, andpertussis vaccines; haemophilus influenzae type b vaccine (Hib);hepatitis A vaccine; hepatitis B vaccine; influenza vaccine; measles,mumps, and rubella vaccines (MMR); pneumococcal vaccine; polio vaccines;rotavirus vaccine; anthrax vaccines; and tetanus and diphtheria vaccine(Td) (see, e.g., U.S. Pat. No. 6,309,633).

In some embodiments, antigens can include any type of antigen associatedwith cancer such as, for example, tumor associated antigens (TSAs)(including antigens associated with leukemias and lymphomas) such ascarcinoembryonic antigen, prostatic acid phosphatase, PSA, PSMA, and thelike, and antigens that are associated with agents that can cause cancer(e.g., tumorigenic viruses such as, for example, adenovirus, HBV, HCV,HTLV, Kaposi's sarcoma-associated herpes virus, HPV (Gardasil), and thelike).

Antigens can include combinations of antigens such as combinations ofpeptides, polysaccharides, lipids, nucleic acids, and the like. Antigenscan include glycoproteins, glycolipids, glycoproteins, lipoproteins,lipopolysaccharides, and the like.

Antigens that are used to carry out the disclosed EP methods includethose that are derivatized or modified in some way, such as byconjugating or coupling one or more additional groups thereto to enhancefunction or achieve additional functions such as targeting or enhanceddelivery thereof, including techniques known in the art such as, forexample, those described in U.S. Pat. No. 6,493,402 to Pizzo et al. (α-2macroglobulin complexes); U.S. Pat. No. 6,309,633; U.S. Pat. No.6,207,157; and U.S. Pat. No. 5,908,629.

Illustrative examples of the MID 100, 200 and the method of using theMID 100, 200 are described in greater detail below.

EXAMPLES Example 1 Effect of Electrode Composition on TransfectionEfficiency

To address the effect of electrode material on reporter genelocalization, transfection efficiency was compared for two minimallyinvasive devices (MID) with different electrode compositions (gold andstainless steel). This comparison was to assess whether a cheaperalternative (stainless steel) to gold electrodes could be used whilestill maintaining transfection efficacy. The electrode composition waseasily tested, because the gold-plated electrodes were easily removedfrom their sockets and replaced with stainless steel electrodes of thesame gauge and length. This produced an identical electrode headdiffering only in the electrode composition.

The experimental outline is detailed in Table 1.

TABLE 1 DNA Number of Biopsy Number of Electrode DNA ConcentrationTreatment Removal Time Animals Composition Delivered (mg/ml) Sites(hours) Required Final Analysis Gold- pgWIZ- 0.5 10 12, 24, 48 4 Grossplated GFP 1 10 12, 24, 48 Visualization/ 2 10 12, 24, 48 HistologyStainless pgWIZ- 0.5 10 12, 24, 48 4 Gross steel GFP 1 10 12, 24, 48Visualization/ 2 10 12, 24, 48 Histology

A series of in vivo expression localization studies were completed. Allin vivo experiments were conducted in Hartley guinea pigs (Charles RiverLaboratories, Wilmington, Mass.), which are considered an excellentmodel for dermatologic applications. All experiments were conductedunder institutional IACUC protocols. All animal experiments wereconducted in accordance with U.S. Department of Defense (DoD) 3216.1“Use of Laboratory Animals in DoD Programs,” 9 CFR parts 1-4 “AnimalWelfare Regulations,” National Academy of Sciences Publication “Guidefor the Care & Use of Laboratory Animals,” as amended, and theDepartment of Agriculture rules implementing the Animal Welfare Act (7U.S.C. 2131-2159), as well as other applicable federal and state lawsand regulations and DoD instructions. All animal treatments were carriedout under anesthesia.

A plasmid expressing the reporter gene GFP was injected intradermally(0.5, 1, and 2 mg/mL) to guinea pig skin. Immediately followinginjection, the skin was electroporated at the injection site using a MIDdevice with either gold or stainless steel electrodes. Animals weresacrificed three days post treatment. Skin was excised and visualizedunder a fluorescent microscope. High resolution photographs were takenand subsequently analyzed for pixel intensity using standard software(Adobe Photoshop CS5). The level of expression was calculated throughpixel counts of pre-defined treatment areas. A “gated region” ofelectrode contact for pixel analysis was established on the presumptionthat transfection occurs only where the electric field is applied andthat the electric field is formed only where the electrodes are indirect contact with the skin. The distance between the first and fourthelectrode in the MID device was 4.5 mm. As such, the ‘ruler tool’ inAdobe Photoshop CS5 was used to isolate a 4.5 mm² region, which wasdefined as approximately 95 pixels in length. Adobe Photoshop CS5recognized pixel intensities ranging from 0-255 (darkest-brightest) inthree different channels (Red, Green, Blue). Since positive GFP signalwould predominate in the green channel, pixel analysis was restricted tothis channel. The CS5 version of Adobe Photoshop was able toautomatically calculate mean and median pixel intensity of the selectedregion. Since the distribution of pixel intensity was not symmetrical inmost cases, the median was deemed to give a better representation ofcentral tendency for the histogram. To ensure accurate results, pooleddata from multiple treatment sites on multiple animals was analyzed.

Results are shown in FIG. 5. Results were also compared to GFPexpression following intradermal injection without subsequentelectroporation. There was no statistically significant differencebetween the gold and stainless steel groups (P value<0.05 between bothtreatment groups and the ID injection-only control). The resultssuggested that the cheaper stainless steel electrode was as effective asthe gold electrode at eliciting reporter gene expression.

Example 2 Effect of Electrode Spacing on Transfection Efficiency

To assess the effect of electrode spacing on transfection efficiency andreporter gene localization, a 1 mm spaced circuit board was created andfitted in a head-piece housing and compared with a similar MID with a1.5 mm spaced circuit board. To ensure that the surface treatment arearemained the same between the two hand pieces at the different spacings,an additional row of electrodes was added to the 1 mm spaced circuitboard. Therefore, a 1.5 mm spacing hand piece with 4×4 rows ofelectrodes (16 electrodes) was compared to a 1 mm spacing hand piecewith 5×5 rows of electrodes (25 electrodes). As such, each hand piecehad an approximate treatment surface area of approximately 4-4.5 mm².FIG. 6A shows a photograph of both hand pieces from a side perspective.The top hand piece is the 1 mm spacing, and the bottom hand piece is the1.5 mm spacing. FIG. 6B shows a close-up view of the face of the 1 mmhand piece.

A series of in vivo expression localization studies were completed, asdescribed in Example 1. Specifically, following an intradermal injectionof a known dose (0.5, 1, and 2 mg/mL) of plasmid DNA expressing thereporter gene GFP into guinea pig skin, the 1 mm or 1.5 mm prototypedevice was used to almost immediately, within 10 seconds afterinjection, electroporate the resulting injection bubble. Electrodes wereeither gold or stainless steel in composition. Animals were sacrificedthree days post treatment. Skin was excised and visualized under afluorescent microscope. High resolution photographs were taken andsubsequently analyzed for pixel intensity using standard software (AdobePhotoshop CS5). The level of expression was calculated through pixelcounts of pre-defined treatment areas. To ensure accurate results,pooled data from multiple treatment sites on multiple animals wasanalyzed. Expression of the GFP was monitored over different timeperiods (12, 24, and 48 hours) to allow assessment of expressionkinetics.

The experimental outline is detailed in Table 2.

TABLE 2 DNA Number of Biopsy Number of Electrode DNA ConcentrationTreatment Removal Time Animals Spacing Delivered (mg/ml) Sites (hours)Required Final Analysis 1 mm pgWIZ- 0.5 10 12, 24, 48 4 Gross GFP 1 1012, 24, 48 Visualization/ 2 10 12, 24, 48 Histology 1.5 mm   pgWIZ- 0.510 12, 24, 48 4 Gross GFP 1 10 12, 24, 48 Visualization/ 2 10 12, 24, 48Histology

Results are shown in FIG. 7. Results are shown for 6 treatments for eachcondition. Results were also compared to GFP expression followingintradermal injection without subsequent electroporation. There was nostatistically significant difference between results for the device withthe 1.5 mm spaced circuit board and the 1 mm spaced circuit board (Pvalue<0.05 between the treatment groups and the ID injection-onlycontrol). The results are representative of multiple experiments anddemonstrated that successful, robust transfection was achieved with aminimally invasive device electroporation (MID EP) using either 1.5 mmor 1 mm electrode spacing.

These results suggested that electrode spacing does not impact GFPexpression in skin because no visible difference (as determined by eyeand quantifiably through pixel counting) was observed between the twoelectrode spacings. Thus, electroporation with MID EP using either 1.5mm or 1 mm electrode spacing resulted in robust reporter geneexpression.

Example 3 Effect of Electrode Spacing on Current

A device as described herein can have the capacity to capture and storeall electrical parameters real time as they occur during eachelectroporation pulse. A series of in vivo expression localizationstudies were completed, as described in Example 1, to examine currentand voltage for electroporation with devices of different electrodespacing and composition. While the applied voltage remained constant (15volts), the impedance (resistance) and current delivered for eachtreatment was examined for each electrode spacing and each electrodecomposition.

FIG. 8 shows both the resulting impedance (resistance in Ohms) andCurrent (in milli Amps).

The current was approximately three times greater in the 1 mm hand piece(average 85 mA) compared to the 1.5 mm hand piece (average 23 mA), yetthe applied voltage was the same across all conditions. The increasedcurrent in the 1.5 mm hand piece resulted in a large reduction(approximately 75%) in the impedance of the tissue. From the perspectiveof producing a tolerable dermal device, increased current can beproblematic by causing more pain or sensation to the patient. Increasedcurrent can cause more pain or sensation in a patient, and thus,increased current can be problematic in producing a tolerable dermaldevice. These results suggested that while the electrode spacing did notappear to impact the resulting GFP expression, either the spacing or thepresence of the additional electrodes can impact the current flow and,as such, impact the impedance of the tissue.

To further address the issue of increased current, it was investigatedwhether the applied voltage could be reduced by a third (to 5 volts) andstill result in 10-20 mA current. Electrodes were stainless steel.Results are shown in FIG. 9. The results suggested that additionalelectrodes, and not the actual electrode spacing, affected the resultingcurrent.

To assess whether a reduced voltage affected the transfection efficacy,a DNA plasmid expressing the reporter gene GFP was deliveredintradermally to guinea pig skin and immediately followed withelectroporation using a MID device with either 1.5 mm (4×4) electrodespacing or 1 mm (4×4 or 5×5) electrode spacing at 15 or 5 volts. Theanimals were sacrificed three days post treatment. The skin was excisedand visualized under a fluorescence microscope. High resolutionphotographs were taken and subsequently analyzed for pixel intensityusing standard software (Adobe Photoshop CS5).

Results are shown in FIG. 10. These results suggested that the inputvoltage can be reduced on larger electrode hand pieces while stillmaintaining transfection efficacy. As such, the results suggested thatrobust transfection can be achieved with a larger array whilemaintaining pain-free and low voltage parameters. It is likely that in ahuman clinical device, at least 25 electrodes would be required tomaintain optimal coverage depending on the usage requirements orpreferences, e.g., the injection volume.

Example 4 Effect of Plasmid Concentration on Transfection Efficiency

The effect of lower concentrations of plasmid expressing the reportergene GFP on transfection efficiency was examined with devices ofdifferent composition and electrode spacing.

A series of in vivo expression localization studies were completed, asdescribed in Example 1. A plasmid expressing the reporter gene GFP wasinjected intradermally (0.5, 0.25, and 0.1 mg/mL) to guinea pig skin andimmediately followed with electroporation using a MID device with eithergold or stainless steel electrodes and either 1 mm- (5×5) or 1.5 mm-(4×4) spaced electrodes at 15 volts. The animals were sacrificed threedays post treatment. The skin was excised and visualized under afluorescence microscope. High resolution photographs were taken andsubsequently analyzed for pixel intensity using standard software (AdobePhotoshop CS5). The level of expression was calculated through pixelcounts of pre-defined treatment areas. To ensure accurate results,pooled data from multiple treatment sites on multiple animals wasanalyzed.

Results are shown in FIG. 11. The GFP expression following ID injectionalone (no EP) was also observed but minimal expression was detected(data not shown). No statistically significant differences betweeneither spacing or electrode composition was observed at any of theconcentrations of the plasmid expressing the reporter gene GFP.

Example 5 Electroporation Efficiency Analyzed at the Cellular Level

Skin samples removed from treated animals from experiments detailed inthe above Examples were analyzed immunohistochemically. Skinpost-treatment was excised post-mortem, sectioned, and paraffin mounted.GFP-expressing cells were observed and counted using a high poweredfluorescent microscope (Olympus—BX51 TF). The number and region (i.e.,layer of strata in epidermis) of GFP-expressing cells were noted.Histological sections were also counter-stained with a collection ofcommercially available antibodies prior to mounting to allow the directidentification of transfected cell types, such as lymphocyte IHC,keratinocytes (the majority of cells in the epidermis), and Langerhanscells (most common APC's in the epidermis). The antibodies were alsoused to observe the effect of electroporation on lymphocyteinfiltration.

Robust keratinocyte staining was achieved in the epidermis. Theepidermal region in the skin sections was clearly defined. Overall, theresults suggested that the additional electrodes massively impacted thecurrent flow and, as such, impacted the impedance of the tissue. Thisincreased current flow did not appear to affect the resulting expressionof the reporter gene. However, it was apparent that the voltage could bereduced on the 5×5 1 mm hand piece while still achieving strong currentsand robust transfection.

To see positive staining for the Langerhans specific antibody in theskin, spleen and lymph nodes were removed from a sacrificed animal touse as positive controls for the antibody. Strong antibody staining wasdetected in both the spleen and the lymph nodes but not in the skin.Although not wishing to be bound by a particular theory, this suggestedthat the antibody was working but that either the signal in the skin wastoo weak to detect, or there were no Langerhans cells present in thetissue tested.

Additional results are shown in FIG. 12. It was observed that bothelectroporation and expression of reporter genes resulted ininfiltration of lymphocytes to the treatment area. The combination of EPand reporter gene expression resulted in the largest infiltration. Whileco-localization of kerotinocyte staining and reporter gene expressionwas observed, the staining was not consistent. However, co-localizationof lymphocyte IHC and reporter gene expression was consistentlyconfirmed.

Example 6 Dual-Head Device

A dual-head device having two arrays side by side with a small bufferzone was manufactured. The arrays were designed to deliver pulsessimultaneously. Alternatively, each head can be pulsed independentlywith additional equipment modifications. Plastic housings and customelectrical components were prototyped. The devices are shown in FIG. 13.

A series of in vivo expression localization studies were completed, asdescribed in Example 1. A plasmid expressing the reporter gene GFP orRFP was injected intradermally at a concentration of 1 mg/mL to guineapig skin. Injection was immediately followed with electroporation usingthe dual head device (with 16 stainless steel electrodes in a 4×4 arrayand a spacing of 1.5 mm, at 25 V). The results are shown in FIG. 14,which is a fluorescent micrograph showing the GFP expression followingan intradermal administration of a reporter gene plasmid and EP with theMID 100, 200, compared to the GFP expression following the intradermalinjection alone (no EP). The electrodes 110 of the MID 100, 200 werespaced from one another by a spacing of 1.5 mm. The animal wassacrificed post treatment and the skin excised and visualized under afluorescence microscope. The fluorescent micrograph confirms thatmultiple plasmids were delivered simultaneously at spatially separatedsites.

Example 7 Kinetics of Transfection

A series of in vivo expression localization studies will be completed,using the methods as described in Example 1. Specifically, following anintradermal injection of a known dose (0.5, 1, and 2 mg/mL) of plasmidDNA expressing the reporter gene GFP into guinea pig skin, a 1 mm or 1.5mm prototype device will be used to immediately electroporate theresulting injection bubble. Electrodes of the MID will be either gold orstainless steel in composition. Animals will be sacrificed at differenttime points (12 hours, 24 hours, and 48 hours, 3 days) post treatment.Skin will be excised and visualized under a fluorescent microscope foreach time point. High resolution photographs will be taken andsubsequently analyzed for pixel intensity using standard software (AdobePhotoshop CS5). The level of expression will be calculated through pixelcounts of pre-defined treatment areas. To ensure accurate results,pooled data from multiple treatment sites on multiple animals will beanalyzed. Expression of the GFP will be compared over the different timeperiods to facilitate assessment of expression kinetics.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made without departing from the spirit and scope of thedisclosure as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this disclosureas defined in the claims appended hereto.

1. A multi electrode array device for electroporating and delivering oneor more antigens to epidermal tissue, the device comprising: a housing;a plurality of electrode arrays projecting from the housing, theelectrode arrays defining spatially separate sites, each electrode arrayincluding at least one electrode; a pulse generator electrically coupledto the electrodes; a programmable microcontroller electrically coupledto the pulse generator; and an electrical power source coupled to thepulse generator and the microcontroller, wherein the electrodes areconfigured to deliver an electroporating pulse to cells of epidermaltissues, and wherein the microcontroller is configured to adjustparameters of the electroporating pulse of each electrode arrayindependently.
 2. The device of claim 1, wherein the electroporatingpulse is associated with an electrical potential, and wherein themicrocontroller is configured to vary the electrical potential fromarray to array.
 3. The device of claim 1, wherein the electroporatingpulse is associated with an electrical current, and wherein themicrocontroller is configured to vary the electrical current from arrayto array.
 4. The device of claim 1, wherein the electroporating pulse isassociated with a duration, and wherein the microcontroller isconfigured to vary the duration from array to array.
 5. The device ofclaim 1, wherein the microcontroller is configured to adjust arespective quantity of electroporating pulses for each electrode arrayindependently, and wherein the quantity is about 1 to about
 10. 6. Thedevice of claim 1, wherein the plurality of electrode arrays are spacedapart from one another at least by a distance so as to substantiallyprevent interference of the antigens when the electroporating pulses aredelivered.
 7. The device of claim 1, wherein the electrodes in eachelectrode array are arranged in a pattern of geometric shape.
 8. Thedevice of claim 1, wherein at least one electrode includes atissue-penetrating end with a length of about 0.1 mm or less.
 9. Thedevice of claim 1, wherein at least one electrode includes atissue-penetrating end with a length of about 0.01 mm to about 0.04 mm.10. The device of claim 1, wherein the electroporating pulses areassociated with an electrical field that substantially prevents damagein the cells of the epidermal tissues.
 11. The device of claim 1,wherein the electroporating pulses are associated with an electricalpotential that is nearly painless as measured by a visual analog scale.12. The device of claim 1, wherein adjoining electrodes are spaced apartfrom one another at a distance of not less than about 1.0 mm.
 13. Thedevice of claim 1, wherein adjoining electrodes are spaced apart fromone another at a distance of not less than about 1.5 mm.
 14. The deviceof claim 1, wherein the electrodes are configured to deliver theelectroporating pulses substantially simultaneously.
 15. A method ofelectroporating and delivering one or more antigens to cells ofepidermal tissues using the device of claim 1, the method comprising:administering the one or more antigens to the cells of the epidermaltissues; contacting the epidermal tissues with the electrodes; anddelivering the electroporating pulses.
 16. The method of claim 15,wherein the contacting step comprises penetrating the epidermal tissuewith at least one electrode at a depth of about 0.1 mm or less.
 17. Themethod of claim 15, wherein the contacting step comprises penetratingthe epidermal tissue with at least one electrode at a depth of about0.01 mm to about 0.04 mm.
 18. The method of claim 15, wherein theelectroporating pulses are associated with an electrical field thatsubstantially prevents damage in the cells of the epidermal tissues. 19.The method of claim 15, wherein the electroporating pulses areassociated with an electrical potential that is nearly painless asmeasured by a visual analog scale.
 20. The method of claim 15, whereinthe electroporating pulses are associated with an electrical potentialof about 1 volts to about 30 volts.
 21. The method of claim 15, whereinthe electroporating pulses are associated with an electrical current ofabout 1 mA to about 50 mA.
 22. The method of claim 15, wherein theelectroporating pulses are associated with a duration ranging from about5 ms to about 250 ms.
 23. The method of claim 15, wherein the deliveringstep comprises delivering a quantity of electroporating pulses, andwherein the quantity is about 1 to about
 10. 24. The method of claim 15,wherein the antigen comprises a nucleic acid, a peptide, or a smallmolecule.
 25. The device of claim 1, wherein one electrode array isspaced apart from an adjacent electrode array by at least a distance soas to substantially prevent interference of multiple antigens deliveredby the arrays, wherein the distance is 1.5 mm.